Purpose or scope: A role for somatic mutations in carcinogenesis and genetic disease is well accepted, but the degree to which mutation rates influence cancer initiation and development is under continuous debate. Recently accumulated genomic data has revealed that thousands of tumor samples are riddled by hypermutation, broadening support that many cancers acquire a mutator phenotype. This major expansion of cancer mutation datasets has provided unprecedented statistical power for the analysis of mutation spectra, which has confirmed several classical sources of mutation in cancer, highlighted new prominent mutation sources and empowered the search for cancer drivers. In our work we combined mechanistic knowledge obtained through our experiments with yeast models to interrogate the large whole-genome datasets of cancer mutations in order to gain mechanistic insight for understanding the impact of mutations on cancer and genetic disease. Research subject: We combined analysis of mutation signatures of human APOBEC3A and APOBEC3B separately expressed in yeast. The mutation signatures defined in the yeast experiments were then applied to interrogation of large datasets of mutations in human cancers Significant materials equipment or methods: Analysis of genome-wide mutation spectra was empowered by a package of flexible analytical tools that can be configured by researchers to test specific hypotheses about mutagenesis patterns within a complex mix of mutagenic processes operating throughout the history of individual cancers. Specific version of this package is set for identifying cancer samples with APOBEC mutagenesis pattern. This analysis, called Pattern of Mutagenesis by APOBEC Cytidine Deaminases (P-MACD), has been recently integrated into the Broad Institute TCGA GDAC Firehose and is currently available for Firehose standard and customized runs. It allows users to explore correlations of APOBEC mutagenesis with multiple clinical and molecular features, e.g., gene expression and hotspots in significantly mutated. Another critical component in our research was expression of human APOBEC enzymes in a yeast reporter strain (deleted for uracil glycosylase) that generates chromosomal ssDNA upon temperature shift. Telomere uncapping in the presence of ssDNA-damaging mutagens results in selectable mutation clusters inactivating multiple reporter genes. Importantly, resection of the complementary strand precludes excision repair and uracils from cytidine deaminations gave rise to C &#8594; T transitions, which marks positions of all cytidine deaminations made by an APOBEC enzyme Accomplishments: Previous indirect evidence implicated APOBEC3B as the more likely major mutator deaminase in human cancers, while APOBEC3As role is not established. Using yeast models enabling controlled generation of long single-strand genomic DNA substrates, we showed the mutation signatures of APOBEC3A and APOBEC3B are statistically distinguishable. We then applied three complementary approaches to identify cancer samples with mutation signatures resembling either APOBEC. Strikingly, APOBEC3A-like samples have over ten-fold more APOBEC-signature mutations than APOBEC3B-like samples. We propose that APOBEC3A mutagenesis is much stronger because APOBEC3A itself is highly proficient at generating DNA breaks, whose repair can trigger formation of single-strand hypermutation substrates.